Immunoblot Analysis of Cellular Proteins

Whole-cell lysates were prepared as previously described [66 (link)]. Protein lysates (25 μg) were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gels, followed by being electrotransferred onto a nitrocellulose filter. The membranes were incubated with primary antibodies, and then with peroxidase-conjugated IgG antibodies (Agilent Technologies, Santa Clara, CA, USA). Immune complexes were detected using an ECL Western blot detection system (Amersham, Aylesbury, UK). The following primary antibodies were used, at a working dilution of 1:1000, for immunoblot analyses: antibodies against CD44 (c-9960), CD47 (sc-7059), nucleostemin (NS) (sc-166460), MDR (sc-13131), RELA (sc-372), β-actin (sc-47778) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), E-cadherin (Abcam, Cambridge, MA, USA; ab1416), snail (#3879), and phospho-Akt (Ser473) (#4060) (Cell Signaling Technology Japan, Tokyo, Japan).